Substance use typically begins in mid-adolescence, but when it occurs earlier in adolescence/late childhood, drug use is associated with significant, life-long addiction liability. Identification of at-risk individuals is vital for early intervention/prevention and may have the greatest impact on reducing addiction long-term. However, this approach requires an understanding of the mechanism of risk and the proof should be causal, and not correlational, as in previous studies. In addition, this understanding needs to be extended to immature females, where extreme little is know about neural changes and drug sensitivity. Here, we propose to build on our substantial preliminary data that utilizes highly novel and innovative approaches to show the following: a) elevated D1 dopamine receptor expression on glutamatergic neurons in the prelimbic prefrontal cortex (plPFC) is observed in adolescent male rats relative to younger and older rats, and is associated with increased sensitivity to drug-associated cues at this age; b) gene engineered elevations in D1 receptors on glutamate neurons with a lentiviral vector (CamKII.D1) in the plPFC increases preferences to cocaine-associated cues; c) gene engineered D1 receptor expression on both GABA and glutamate neurons (Synapsin.D1) does not enhance drug-cue sensitivity; d) novelty-seeking in juvenile rats correlates with preferences for cocaine-associated environments (r=0.87); e) D1 dopamine receptors produce age-dependent changes in blood flow patterns that most likely reflect the distribution of D1 receptors on glutamate and GABA neurons. In a set of studies that are highly consistent with # PA-07-226, we propose to use these two viral vectors to increase (a) or decrease (b) sensitivity to drug-related cues to experimentally show that D1 receptor location on glutamate neurons is the risk mechanism (Aim I). These same animals will also show that the original, strong drug-cue associations are more difficult to extinguish and reinstate more strongly as a result of D1 receptors on glutamate neurons within the plPFC (Aim I). Aim II will determine whether novelty-seeking in juvenile animals can serve as an behavioral metric to identify increased drug-cue sensitivity and how this relates to D1 receptors. Aim IV, which runs concurrently, will use the CamKII.D1 virus and novelty-seeking to determine whether pharmacoMRI can identify this biomarker of risk through unique blood flow patterns. Aim III will determine whether a more benign environment at the time of initial drug exposure (e.g., home- cage) can reduce drug-seeking in other more salient environments later in life. Together, these studies will determine how a single biomarker predicts risk for substance use and influence enduring risk of drug abuse. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to determine whether elevated expression of the D1 dopamine receptor in the prefrontal cortex contributes to vulnerability to substance use in developing animals and can serve as a biomarker for early detection. D1 dopamine receptors are transiently over-expressed on specific neurons (e.g., glutamate) during adolescence, which is hypothesized to underlie enhanced vulnerability to cocaine at this stage. Here, we will use gene engineering to manipulate D1 receptor expression to conclusively show that this receptor underlies increased sensitivity to the initial formation of cocaine-associations and their maintenance, which may be detected by behavioral screening early and/or detected with pharmacoMRI and associated blood flow changes.